Human Soluble Secreted Endopeptidase (Human SEP)
Proteases form a large family of enzymes that cleave proteins and peptides at the peptide bond that forms the backbone of the peptide or protein chain. Proteases are found in all organisms from bacteria to man. In humans approximately 1% of all genes (400-1000) are predicted to encode protease enzymes. They participate in activation and maturation of nascent polypeptides, the degradation of mis-folded and damaged proteins, and the controlled turnover of peptides and proteins both inside, and outside, the cell. Their activities are important for many processes including digestion, normal growth, endocrine function, wound healing, inflammation, angiogenesis, tissue remodelling during embryonic development, tumour metastasis, cardiovascular disease, neurological disease, and bacterial, parasitic, and viral infections.
Proteases can be broadly categorised on the basis of where they cleave their substrate. Exopeptidases, which include aminopeptidases, dipeptidyl peptidases, tripeptidases, carboxypeptidases, peptidyl-di-peptidases, dipeptidases and omega peptidases, cleave residues at the termini of their substrate. Endopeptidases, including serine proteases, cysteine proteases, and metalloendopeptidases, cleave at a sequence within the peptide.
An important group of endopeptidases known as zinc metalloproteases are characterised by having a requirement for the binding of a zinc ion in their catalytic site. Zinc metalloproteases can be subdivided into classes (for review see FEBS Letters 354 (1994) pp. 1-6), with one such class being the neprilysin (NEP)-like zinc metalloproteases (FASEB Journal, Vol 11, 1997 pp. 355-384). The NEP class includes at least 7 enzymes that are structurally related to each other (see later). They are typically membrane-bound, with a large carboxy-terminal extracellular domain, a short membrane-spanning region, and a short intracellular domain at the amino terminus. Known members of this family are neprilysin (also called NEP, CD10, CALLA, enkephalinase or EC 3.4.24.11), endothelin-converting enzymes (ECE-1 and ECE-2), PEX, KELL, X-converting enzyme/damage induced neural endopeptidase (XCE/DINE), and an enzyme identified in rodents called soluble secreted endopeptidase/neprilysin II (SEP/NEPII; Ghaddar, G et al, Biochem Journal, Vol 347, 2000, pp. 419-429; Ikeda, K et al, Journal Biological Chemistry, Vol 274, 1999, pp. 32469-32477; Tanja, O et al, Biochem Biophys Research Communication, Vol 271, 2000, pp. 565-570; International Patent Application WO 99/53077). The functions of the members of this class are thought to be related to peptidergic signalling. This is a process that occurs in most organisms, including humans, in which peptide molecules are used as “messengers” to elicit physiological responses. This typically involves the production and release of the peptide messenger by a specific cell, sometimes as an inactive precursor that is cleaved by a protease to become active. The active form of the peptide then binds a specific receptor on the surface of another cell where it elicits a response. The peptide is then inactivated by degradation by another protease.
NEP was the first member of this class to be discovered. NEP is a promiscuous protease in that it is able to proteolyse and inactivate many biological peptides, e.g. enkephalins, bradykinin and substance P. It usually cleaves the peptide on the amino-terminal side of a hydrophobic residue. NEP can be found in many tissues of the body and is most abundant in kidney. Its physiological functions are not fully understood, but one indication from the phenotype of NEP “knockout” mice is that it is involved in preventing endotoxic shock.
ECE-1 is a protein 37% identical to NEP. ECE-1 is broadly distributed throughout the body and converts the inactive precursor peptide big-endothelin into endothelin, which is a potent vasoconstrictor, important for maintaining vascular tone. The ECE-2 enzyme is derived form a separate gene to ECE-1, but its amino acid sequence is similar, with an overall homology of 59%. The physiological importance of ECE-2 to endothelin production is unclear. ECE-2 mRNA is present at much lower levels then ECE-1 mRNA and it has a different pH optimum to ECE-1, being inactive at neutral pH, and most active at pH 5.5.
The KELL enzyme is a clinically important member of the NEP class found in erythroid tissues. The antigens of the KELL blood group system reside in this protein which can cause haemolytic reaction to blood transfusions.
The PEX gene was identified as being genetically linked to a disorder called X-linked hypophosphatemic rickets, a dominant disorder typified by decreased renal tubular phosphate reabsorbtion. Based on its close homology to the other members of the NEP family (49-60%) it is also predicted to be a membrane-bound metalloprotease, but no substrate has yet been found.
XCE (Valdenaire, O et al, Molecular Brain Research, Vol 64, 1999, pp. 211-221), and its rat equivalent DINE (Kiryu-Seo, S et al, Proceedings of the National Academy of Science USA, 2000, pp. 4345-4350), are expressed predominantly in the central nervous system. DINE expression is up-regulated following neuronal damage, and this is thought to help prevent neuronal apoptosis, possibly as a result of the DINE-mediated proteolytic activation of antioxidant enzymes. A physiological substrate of XCE/DINE has also not yet been identified, but from their sequence they are clearly predicted to be proteases, and for DINE, this has been proven using a synthetic peptide substrate.
Rodent SEP and NEPII were discovered most recently. NEPII is likely to be a rat equivalent of SEP, which is a mouse enzyme, as they share 91% amino acid identity. They are the members of this class closest to NEP in their amino acid sequence, both being 54% identical to human NEP. The mRNA of both is highly abundant in the testis and can also be detected at low levels in a broad range of other tissues. In the case of rat NEPII, the mRNA has also been found at comparatively high levels in the brain and pituitary. When produced recombinantly in mammalian cells, both mouse SEP and rat NEPII can be found in the growth media. This suggests they could be secreted proteases that may be able to circulate and hence cleave peptides at other sites in the body. Mouse SEP and rat NEPII, like some other members of this class such as ECE-1, exhibit splice variation. In the case of mouse SEP and rat NEPII, this splice variation results in isoforms with alterations in sequences involved in membrane localisation and secretion. The physiological significance of this is unclear but it is likely there could be membrane-bound, circulating, and intracellular forms of these enzymes. Mouse SEP has been shown to be able to cleave a range of important biological peptides including enkephalin, endothelin, big-endothelin, Bradykinin and substance P. Like NEP, therefore, it has a fairly broad substrate specificity and may have several physiological functions in different tissues.
Enzymes in this NEP class, like other metalloprotease enzymes, have been shown to be amenable to inhibition by small drug-like molecules (for example, thiorphan and phosphoramidon). This, together with the emerging nature of the physiological function of some members of the NEP-like enzymes in modulating peptidergic signalling, makes them attractive targets for pharmaceutical intervention. NEP inhibitors have been developed for indications including cardiovascular disease, and it is likely that, as knowledge of their function increases further, specific inhibitors of NEP-like enzymes may have a role in the prophylaxis and/or treatment of many other indications such as sexual dysfunction (especially male sexual dysfunction, e.g. male erectile dysfunction (MED), and female sexual dysfunction (FSD), e.g. female sexual arousal disorder (FSAD)), preterm labour, pre-eclampsia, endometriosis, reproductive disorders (especially male and female infertility, polycystic ovarian syndrome, implantation failure), hypertension, heart failure, angina, renal insufficiency, cyclical oedema, hyperaldosteroneism, glaucoma, asthma, inflammation, leukemia, pain, epilepsy, affective disorders, dementia and geriatric confusion, obesity and gastrointestinal disorders (especially diarrhoea and irritable bowel syndrome), septic shock, the modulation of gastric acid secretion and the treatment of hyperreninaemia.
Sexual Dysfunction
Sexual dysfunction (SD) is a significant clinical problem which can affect both males and females. The causes of SD may be both organic as well as psychological. Organic aspects of SD are typically caused by underlying vascular diseases, such as those associated with hypertension or diabetes mellitus, by prescription medication and/or by psychiatric disease such as depression. Physiological factors include fear, performance anxiety and interpersonal conflict. SD impairs sexual performance, diminishes self-esteem and disrupts personal relationships thereby inducing personal distress. In the clinic, SD disorders have been divided into female sexual dysfunction (FSD) disorders and male sexual dysfunction (MSD) disorders (Melman et al 1999). FSD is best defined as the difficulty or inability of a woman to find satisfaction in sexual expression. Male sexual dysfunction (MSD) is generally associated with erectile dysfunction, also known as male erectile dysfunction (MED) (Benet et al 1994).
The compounds of the invention are particularly beneficial for the prophylaxis and/or treatment of sexual dysfunction in the male (e.g. male erectile dysfunction—MED) and in the female—female sexual dysfunction (FSD), e.g. female sexual arousal disorder (FSAD).
Female Sexual Dysfunction (FSD)
In accordance with the invention, FSD can be defined as the difficulty or inability of a woman to find satisfaction in sexual expression. FSD is a collective term for several diverse female sexual disorders (Leiblum, S. R. (1998). Definition and classification of female sexual disorders. Int. J. Impotence Res., 10, S104-S106; Berman, J. R., Berman, L. & Goldstein, I. (1999). Female sexual dysfunction: Incidence, pathophysiology, evaluations and treatment options. Urology, 54, 385-391.). The woman may have lack of desire, difficulty with arousal or orgasm, pain with intercourse or a combination of these problems. Several types of disease, medications, injuries or psychological problems can cause FSD. Treatments in development are targeted to treat specific subtypes of FSD, predominantly desire and arousal disorders.
The categories of FSD are best defined by contrasting them to the phases of normal female sexual response: desire, arousal and orgasm (Leiblum, S. R. (1998). Definition and classification of female sexual disorders. Int. J. Impotence Res., 10, S104-S106). Desire or libido is the drive for sexual expression. Its manifestations often include sexual thoughts either when in the company of an interested partner or when exposed to other erotic stimuli. Arousal is the vascular response to sexual stimulation, an important component of which is genital engorgement and includes increased vaginal lubrication, elongation of the vagina and increased genital sensation/sensitivity. Orgasm is the release of sexual tension that has culminated during arousal.
Hence, FSD occurs when a woman has an inadequate or unsatisfactory response in any of these phases, usually desire, arousal or orgasm. FSD categories include hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorders and sexual pain disorders. Although the compounds of the invention will improve the genital response to sexual stimulation (as in female sexual arousal disorder), in doing so it may also improve the associated pain, distress and discomfort associated with intercourse and so treat other female sexual disorders.
Thus, in accordance with a preferred aspect of the invention, there is provided use of a compound of the invention in the preparation of a medicament for the treatment or prophylaxis of hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder and sexual pain disorder, more preferably for the treatment or prophylaxis of sexual arousal disorder, orgasmic disorder, and sexual pain disorder, and most preferably in the treatment or prophylaxis of sexual arousal disorder.
Hypoactive sexual desire disorder is present if a woman has no or little desire to be sexual, and has no or few sexual thoughts or fantasies. This type of FSD can be caused by low testosterone levels, due either to natural menopause or to surgical menopause. Other causes include illness, medications, fatigue, depression and anxiety.
Female sexual arousal disorder (FSAD) is characterised by inadequate genital response to sexual stimulation. The genitalia do not undergo the engorgement that characterises normal sexual arousal. The vaginal walls are poorly lubricated, so that intercourse is painful. Orgasms may be impeded. Arousal disorder can be caused by reduced oestrogen at menopause or after childbirth and during lactation, as well as by illnesses, with vascular components such as diabetes and atherosclerosis. Other causes result from treatment with diuretics, antihistamines, antidepressants e.g. selective serotonin re-uptake inhibitors (SSRIs) or antihypertensive agents.
Sexual pain disorders (includes dyspareunia and vaginismus) is characterised by pain resulting from penetration and may be caused by medications which reduce lubrication, endometriosis, pelvic inflammatory disease, inflammatory bowel disease or urinary tract problems.
The prevalence of FSD is difficult to gauge because the term covers several types of problem, some of which are difficult to measure, and because the interest in treating FSD is relatively recent. Many women's sexual problems are associated either directly with the female ageing process or with chronic illnesses such as diabetes and hypertension.
Because FSD consists of several subtypes that express symptoms in separate phases of the sexual response cycle, there is not a single therapy. Current treatment of FSD focuses principally on psychological or relationship issues. Treatment of FSD is gradually evolving as more clinical and basic science studies are dedicated to the investigation of this medical problem. Female sexual complaints are not all psychological in pathophysiology, especially for those individuals who may have a component of vasculogenic dysfunction (e.g. FSAD) contributing to the overall female sexual complaint. There are at present no drugs licensed for the treatment of FSD. Empirical drug therapy includes oestrogen administration (topically or as hormone replacement therapy), androgens or mood-altering drugs such as buspirone or trazodone. These treatment options are often unsatisfactory due to low efficacy or unacceptable side effects.
Since interest is relatively recent in treating FSD pharmacologically, therapy consists of the following: psychological counselling, over-the-counter sexual lubricants, and investigational candidates, including drugs approved for other conditions. These medications consist of hormonal agents, either testosterone or combinations of oestrogen and testosterone and more recently vascular drugs, that have proved effective in male erectile dysfunction (MED). None of these agents has yet been demonstrated to be effective in treating FSD.
As discussed, the compounds of the invention are particularly useful for the prophylaxis and/or treatment of female sexual arousal disorder (FSAD).
The Diagnostic and Statistical Manual (DSM) IV of the American Psychiatric Association defines Female Sexual Arousal Disorder (FSAD) as being:    “ . . . a persistent or recurrent inability to attain or to maintain until completion of the sexual activity adequate lubrication-swelling response of sexual excitement. The disturbance must cause marked distress or interpersonal difficulty . . . ”.
The arousal response consists of vasocongestion in the pelvis, vaginal lubrication and expansion and swelling of the external genitalia. The disturbance causes marked distress and/or interpersonal difficulty. Studies investigating sexual dysfunction in couples reveals that up to 76% of women have complaints of sexual dysfunction and that 30-50% of women in the USA experience FSD (Berman, J. R., Berman, L. A., Werbin, T. J. et al. (1999). Female sexual dysfunction: Anatomy, physiology, evaluation and treatment options. Curr Opin Urology, 9, 563-568).
FSAD is a highly prevalent sexual disorder affecting pre-, peri- and post-menopausal (± hormone replacement therapy (HRT)) women. It is associated with concomitant disorders such as depression, cardiovascular diseases, diabetes and urogenital (UG) disorders.
The primary consequences of FSAD are lack of engorgement/swelling, lack of lubrication and lack of pleasurable genital sensation. The secondary consequences of FSAD are reduced sexual desire, pain during intercourse and difficulty in achieving an orgasm.
It has recently been hypothesised that there is a vascular basis for at least a proportion of patients with symptoms of FSAD (Goldstein et al., Int. J. Impot. Res., 10, S84-S90,1998) with animal data supporting this view (Park et al., Int. J. Impot. Res., 9, 27-37, 1997).
Drug candidates for treating FSAD, which are under investigation for efficacy, are primarily erectile dysfunction therapies that promote circulation to the male genitalia. They consist of two types of formulation, oral or sublingual medications (Apomorphine, Phentolamine, phosphodiesterase type 5 inhibitors e.g. Sildenafil), and prostaglandin (PGE1) that are injected or administered transurethrally in men, and topically to the genitalia in women. However, none of these therapies have yet been shown to be effective in the treatment of FSAD.
Without being bound by theory, we believe that neuropeptides such as vasoactive intestinal peptide (VIP) are major neurotransmitter candidates in the control of the female sexual arousal response, especially in the control of genital blood flow. VIP and other neuropeptides are degraded/metabolised by SEP. Thus, SEP inhibitors will potentiate the endogenous vasorelaxant effect of VIP released during arousal. This will lead to a prophylaxis and/or treatment of FSAD, such as through enhanced genital blood flow and thence genital engorgement. We have shown that inhibitors of SEP enhance pelvic nerve-stimulated and VIP-induced increases in vaginal and clitoral blood flow. In addition, we have shown that SEP inhibitors enhance VIP and nerve-mediated relaxations of isolated vagina wall.
Thus the present invention is advantageous as it helps provide a means for restoring a normal sexual arousal response—namely increased genital blood flow leading to vaginal, clitoral and labial engorgement. This will result in increased vaginal lubrication via plasma transudation, increased vaginal compliance and increased genital sensitivity. Hence, the present invention provides a means to restore, or potentiate, the normal sexual arousal response.
By female genitalia herein we mean: “The genital organs consist of an internal and external group. The internal organs are situated within the pelvis and consist of ovaries, the uterine tubes, uterus and the vagina. The external organs are superficial to the urogenital diaphragm and below the pelvic arch. They comprise the mons pubis, the labia majora and minora pudendi, the clitoris, the vestibule, the bulb of the vestibule, and the greater vestibular glands” (Gray's Anatomy, C.D. Elemente, 13th American Edition).
R. J. Levin teaches us that because “ . . . male and female genitalia develop embryologically from the common tissue anlagen, [that] male and female genital structures are argued to be homologues of one another. Thus the clitoris is the penile homologue and the labia homologues of the scrotal sac . . . .” (Levin, R. J. (1991), Exp. Clin. Endocrinol., 98, 61-69).
Male Erectile Dysfunction (MED)
It is known that some individuals can suffer from male erectile dysfunction (MED).
Male erectile dysfunction (MED) is defined as:    “ . . . the inability to achieve and/or maintain a penile erection for satisfactory sexual performance (NIH Consensus Development Panel on Impotence, 1993) . . . ”.
It has been estimated that the prevalence of erectile dysfunction (ED) of all degrees (minimal, moderate and complete impotence) is 52% in men 40 to 70 years old, with higher rates in those older than 70 (Melman et al 1999). The condition has a significant negative impact on the quality of life of the patient and their partner, often resulting in increased anxiety and tension which leads to depression and low self esteem. Whereas two decades ago, MED was primarily considered to be a psychological disorder (Benet et al 1994), it is now known that for the majority of patients there is an underlying organic cause. As a result, much progress has been made in identifying the mechanism of normal penile erection and the pathophysiology of MED.
Penile erection is a haemodynamic event which is dependent upon the balance of contraction and relaxation of the corpus cavernosal smooth muscle and vasculature of the penis (Lerner et al 1993). Corpus cavernosal smooth muscle is also referred to herein as corporal smooth muscle or in the plural sense corpus cavernosa. Relaxation of the corpus cavernosal smooth muscle leads to an increased blood flow into the trabecular spaces of the corpus cavernosa, causing them to expand against the surrounding tunica and compress the draining veins. This produces a vast elevation in blood pressure which results in an erection (Naylor, 1998).
The changes that occur during the erectile process are complex and require a high degree of co-ordinated control involving the peripheral and central nervous systems, and the endocrine system (Naylor, 1998). Corporal smooth muscle contraction is modulated by sympathetic noradrenergic innervation via activation of postsynaptic α1 adrenoceptors. MED may be associated with an increase in the endogenous smooth muscle tone of the corpus cavernosum. However, the process of corporal smooth muscle relaxation is mediated partly by non-adrenergic, non-cholinergic (NANC) neurotransmission. There are a number of other NANC neurotransmitters found in the penis, other than nitric oxide (NO), such as calcitonin gene related peptide (CGRP) and vasoactive intestinal peptide (VIP). The main relaxing factor responsible for mediating this relaxation is NO, which is synthesised from L-arginine by nitric oxide synthase (NOS) (Taub et al 1993; Chuang et al 1998). It is thought that reducing corporal smooth muscle tone may aid NO to induce relaxation of the corpus cavernosum. During sexual arousal in the male, NO is released from neurones and the endothelium and binds to and activates soluble guanylate cyclase (sGC) located in the smooth muscle cells and endothelium, leading to an elevation in intracellular cyclic guanosine 3′,5′-monophosphate (cGMP) levels. This rise in cGMP leads to a relaxation of the corpus cavernosum due to a reduction in the intracellular calcium concentration ([Ca2+]i), via unknown mechanisms thought to involve protein kinase G activation (possibly due to activation of Ca2+ pumps and Ca2+-activated K+ channels; Chuang et al., 1998).
Sildenafil citrate (also known as Viagra™) has recently been developed by Pfizer as the first oral drug treatment for MED. Sildenafil acts by inhibiting cGMP breakdown in the corpus cavernosa by selectively inhibiting phosphodiesterase 5 (PDE5), thereby limiting the hydrolysis of cGMP to 5′GMP (Boolel et al., 1996; Jeremy et al., 1997) and thereby increasing the intracellular concentrations of cGMP and facilitating corpus cavernosal smooth muscle relaxation.
Currently, all other available MED therapies on the market, such as treatment with prostaglandin based compounds i.e. alprostadil which can be administered intra-urethrally (available from Vivus Inc., as Muse™) or via small needle injection (available from Pharmacia & Upjohn, as Caverject™), are either inconvenient and/or invasive. Other treatments include vacuum constriction devices, vasoactive drug injection or penile prostheses implantation (Montague et al., 1996). Although injectable vasoactive drugs show high efficacy, side effects such as penile pain, fibrosis and priapism are common, and injection therapy is not as convenient as oral therapy therefore sildenafil currently represents the most preferred therapy on the market.
There is no prior documented evidence for the expression or a functional role of SEP in the penis or corpus cavernosum or in the erectile mechanism/process.
There is also no prior documented evidence for a functional or biochemical effect for SEP inhibitors on the penis or corpus cavernosum or alternatively in the erectile mechanism/process.
There is no prior documented evidence for the expression or a functional role of SEP in the vagina or clitoral corpus cavernosum or in the female sexual arousal mechanism/process.
There is also no prior documented evidence for a functional or biochemical effect for SEP inhibitors in the vagina or clitoral corpus cavernosum or in the female sexual arousal mechanism/process.
Thus, a seminal finding of the present invention is the ability to treat an male or a female suffering from sexual dysfunction, in particular MED or FSAD, with use of a soluble secreted endopeptidase inhibitor (SEPi). Surprisingly the applicants have also found that inhibition of SEP with a SEPi significantly enhances the nerve-stimulated arousal process.
The present invention is advantageous as it provides a means for restoring a normal sexual arousal response—namely increased penile blood flow leading to erection of the penis in males and a increased vaginal clitoral blood flow leading to genital engorgement in females. Hence, the present invention provides a means to restore, or potentiate, the normal sexual arousal response.
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